CN109406681B - Construction method and detection method of UPLC characteristic spectrum of radix angelicae medicinal material - Google Patents
Construction method and detection method of UPLC characteristic spectrum of radix angelicae medicinal material Download PDFInfo
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Abstract
The invention relates to a construction method and a detection method of a UPLC (ultra performance liquid chromatography) characteristic spectrum of a radix angelicae medicinal material. The method comprises the following steps: preparation of reference solutions: weighing imperatorin reference substance, and dissolving with solvent to obtain reference substance solution; preparation of a test solution: weighing radix Angelicae Dahuricae, adding extraction solvent, performing ultrasonic or reflux extraction, filtering, and collecting filtrate to obtain the sample solution; establishing a characteristic spectrum: respectively sucking the reference substance solution and the test solution, injecting the reference substance solution and the test solution into an ultra-high performance liquid chromatograph, and measuring to obtain the characteristic map; wherein the ultra-high performance liquid chromatography adopts the following elution conditions: acetonitrile is taken as a mobile phase A, and acetic acid water solution with volume concentration of 0.05-0.15% is taken as a mobile phase B; gradient elution was used. The characteristic spectrum established by the method can comprehensively reflect the characteristics of the angelica dahurica medicinal material, and the method is accurate and reliable, good in repeatability and simple and convenient to operate.
Description
Technical Field
The invention relates to the quality research of traditional Chinese medicinal materials, in particular to a construction method and a detection method of a UPLC (ultra performance liquid chromatography) characteristic spectrum of an angelica dahurica medicinal material.
Background
Radix Angelicae Dahuricae is dried root of Angelica dahurica (Fisch. ex Hoffm.) Benth.et hook.f. or Angelica dahurica (Fisch. ex Hoffm.) Benth.et hook.f. var.fortusana (Boiss.) Shann et Yuan. It is originally recorded in Shen nong Ben Cao Jing (Shen nong's herbal Jing), pungent and warm, entering stomach, large intestine and lung meridians, has the actions of relieving exterior syndrome and dispelling cold, dispelling wind and relieving pain, freeing nasal orifices, eliminating dampness and stopping leukorrhagia, and decreasing swelling and expelling pus, and is often used for treating cold, headache, supra-orbital bone pain, nasal obstruction and running nose, allergic rhinitis, nasosinusitis, toothache, leukorrhagia, sore and ulcer, swelling and pain, etc.
The main chemical components and the drug effect components of the angelica dahurica are flavonoids and derivatives thereof and volatile oil which are reported in the literature. Modern pharmacological research shows that the angelica dahurica has the effects of relieving fever, easing pain, resisting inflammation, resisting bacteria, resisting oxidation, resisting aging, protecting heart vessels, resisting tumors and the like. The water extract of radix Angelicae Dahuricae has strong antipyretic effect, the coumarins are the main components of radix Angelicae Dahuricae with antipyretic, analgesic and antiinflammatory effects, and the combined use of radix Angelicae Dahuricae total coumarins and volatile oil can significantly enhance analgesic and antiinflammatory effects. Imperatorin can inhibit lipolysis induced by toxic hormone-L and expression of HIF-1 target gene, thereby playing an anti-tumor role, eliminating depression-like behavior by inhibiting lipolysis induced by toxic hormone-L, and has obvious stimulation effect on 5-HT.
The traditional Chinese medicine is formed by superposing a plurality of components aiming at a plurality of targets, the current quality control standard takes a certain specific component as an index, and along with the modernization of the traditional Chinese medicine research, the quality research of the traditional Chinese medicine gradually shifts from the qualitative and quantitative analysis of a single effective component or an index component to the analysis of an effective component group.
The quality control of angelica dahurica in the Chinese pharmacopoeia 2015 edition is only limited to the content determination of imperatorin. Therefore, the quality control of the angelica dahurica medicinal material is more beneficial to comprehensively mastering the quality of the medicinal material by establishing a characteristic spectrum analysis method of an effective component group besides establishing a content determination method of the effective component imperatorin and establishing a content determination limit. The Chinese medicine characteristic map technology has very important practical significance for increasing the quality control content of Chinese medicines, improving the overall level of the Chinese medicine industry and realizing the trend of Chinese medicines to the world.
In the traditional method, the characteristic spectrum research of the angelica dahurica mainly relates to methods such as HPLC, GC, RRLC and the like, research objects mainly relate to volatile oil and coumarin chemical components, the research on flavonoid components is less, and the established characteristic spectrum has fewer common peaks.
Based on the above, in order to better evaluate the quality of the radix angelicae, a more comprehensive radix angelicae characteristic map is established, and particularly, a quality evaluation system for flavonoid components in the radix angelicae is necessary.
Disclosure of Invention
Therefore, a method for constructing a UPLC characteristic map of the radix angelicae medicinal material is needed. The characteristic spectrum established by the method has 16 common peaks, wherein the characteristic spectrum comprises 9 clear flavonoid components, the characteristics of the radix angelicae medicinal material can be comprehensively reflected, and the method is accurate and reliable, good in repeatability and simple and convenient to operate.
A method for constructing a UPLC characteristic spectrum of a radix angelicae medicinal material comprises the following steps:
preparation of reference solutions: weighing imperatorin reference substance, and dissolving with solvent to obtain reference substance solution;
preparation of a test solution: weighing radix Angelicae Dahuricae, adding extraction solvent, performing ultrasonic or reflux extraction, filtering, and collecting filtrate to obtain the sample solution; the extraction solvent is methanol, methanol water solution with volume concentration of 45-55%, ethanol or ethanol water solution with volume concentration of 45-55%;
establishing a characteristic spectrum: respectively sucking the reference substance solution and the test solution, injecting the reference substance solution and the test solution into an ultra-high performance liquid chromatograph, and measuring to obtain the characteristic map; the characteristic map comprises characteristic peaks of hydrated oxypeucedanin, byak, 8-methoxypsoralen, bergapten, byak brain, oxypeucedanin, imperatorin, phellopterin and isoimperatorin;
wherein the ultra-high performance liquid chromatography adopts the following elution conditions: acetonitrile is taken as a mobile phase A, and acetic acid water solution with volume concentration of 0.05-0.15% is taken as a mobile phase B; gradient elution was used.
In one embodiment, the gradient elution method comprises:
the volume percent of the mobile phase A is increased to 28 percent from 15 percent and the volume percent of the mobile phase B is reduced to 72 percent from 85 percent in 0-5.5 min;
5.5-7min, the volume percent of the mobile phase A is increased from 28% to 40%, and the volume percent of the mobile phase B is decreased from 72% to 60%;
7-9.5min, and keeping the volume percent of the mobile phase A at 40% and the volume percent of the mobile phase B at 60%;
9.5-15.5min, the volume percent of the mobile phase A is increased from 40% to 65%, and the volume percent of the mobile phase B is decreased from 60% to 35%;
15.5-15.51min, the volume percentage of the mobile phase A is reduced from 65% to 15%, and the volume percentage of the mobile phase B is reduced from 35% to 85%;
15.51-18min, keeping the volume percentage of the mobile phase A at 15% and the volume percentage of the mobile phase B at 85%.
In one embodiment, the extraction solvent is 45-55% ethanol water solution, and the amount of the extraction solvent is 10-50mL per 0.5g of radix Angelicae Dahuricae medicinal material; the extraction time is 20-40 min.
In one embodiment, the conditions of the ultra-high performance liquid chromatography are as follows: octadecylsilane chemically bonded silica is used as a filler for the chromatographic column; the flow rate of the mobile phase is 0.25-0.4 mL/min; the column temperature is 30-40 ℃; the detection wavelength is 300nm or 254 nm.
In one embodiment, the reference solution is prepared with methanol as the solvent and the imperatorin is present in the reference solution at a concentration of 8-12 μ g/mL.
In one embodiment, the characteristic map comprises 16 characteristic peaks, the reference peak S is a chromatographic peak of 13 imperatorin, and the relative retention time of each characteristic peak is within ± 10% of a specified value, wherein the specified value is: peak 1: 0.19, peak 2: 0.26, peak 3: 0.35, peak 4: 0.43, peak 5 (hydrated oxypeucedanin): 0.49, peak 6 (byakangenin): 0.51, peak 7: 0.58, peak 8 (8-methoxypsoralen): 0.61, peak 9 (bergamoolide): 0.67, peak 10: 0.71, peak 11 (white angelica brain): 0.79, peak 12 (oxypeucedanin): 0.81, peak 13 (imperatorin): 1. peak 14 (phellopterin): 1.05, peak 15 (isoimperatorin): 1.11, peak 16: 1.12.
a detection method of radix angelicae comprises the following steps:
preparing a sample solution to be tested: weighing radix Angelicae Dahuricae to be tested, adding extraction solvent, performing ultrasonic or reflux extraction, filtering, and collecting filtrate to obtain the sample solution; the extraction solvent is methanol, methanol water solution with volume concentration of 45-55%, ethanol or ethanol water solution with volume concentration of 45-55%;
and (3) detection: absorbing the sample solution to be detected, injecting the sample solution into an ultra-high performance liquid chromatograph, and detecting;
wherein the ultra-high performance liquid chromatography adopts the following elution conditions: acetonitrile is taken as a mobile phase A, and acetic acid water solution with volume concentration of 0.05-0.15% is taken as a mobile phase B; gradient elution was used.
In one embodiment, the gradient elution method comprises:
the volume percent of the mobile phase A is increased to 28 percent from 15 percent and the volume percent of the mobile phase B is reduced to 72 percent from 85 percent in 0-5.5 min;
5.5-7min, the volume percent of the mobile phase A is increased from 28% to 40%, and the volume percent of the mobile phase B is decreased from 72% to 60%;
7-9.5min, and keeping the volume percent of the mobile phase A at 40% and the volume percent of the mobile phase B at 60%;
9.5-15.5min, the volume percent of the mobile phase A is increased from 40% to 65%, and the volume percent of the mobile phase B is decreased from 60% to 35%;
15.5-15.51min, the volume percentage of the mobile phase A is reduced from 65% to 15%, and the volume percentage of the mobile phase B is reduced from 35% to 85%;
15.51-18min, keeping the volume percentage of the mobile phase A at 15% and the volume percentage of the mobile phase B at 85%.
In one embodiment, the extraction solvent is 45-55 vol% ethanol water solution, and the amount of the extraction solvent is 10-50mL per 0.5g radix Angelicae Dahuricae medicinal material.
In one embodiment, the extraction time is 20-40 min.
In one embodiment, the conditions of the ultra-high performance liquid chromatography are as follows: octadecylsilane chemically bonded silica is used as a filler for the chromatographic column; the flow rate of the mobile phase is 0.25-0.4 mL/min; the column temperature is 30-40 ℃; the detection wavelength is 300nm or 254 nm.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention adopts ultra-high performance liquid chromatography (UPLC) method, reasonably controls the chromatographic conditions, takes imperatorin as a reference peak, acetonitrile as a mobile phase A and acetic acid aqueous solution with volume concentration of 0.05-0.15% as a mobile phase B for gradient elution, totally determines 16 characteristic peaks, wherein 9 flavonoid component characteristic peaks with definite chemical structure form the complete picture of the characteristic spectrum of the angelica dahurica medicinal material, so that the quality control of the angelica dahurica medicinal material is upgraded from the original determination of a certain component content or the determination of a few common peaks to the inherent quality detection of the whole angelica dahurica medicinal material, especially the detection of the flavonoid component, can comprehensively reflect the characteristics of the angelica dahurica medicinal material, avoids the defects of single or few chemical component detection, and can be used for determining the chemical components in the angelica dahurica and the identification of the angelica dahurica medicinal material.
(2) The method is simple, has good reproducibility, is accurate and reliable, has simple UPLC gradient elution condition, is convenient to operate, and has the advantages of time saving and less solvent consumption compared with the method for determining the radix angelicae characteristic spectrum by an HPLC method.
(3) According to the specific characteristic spectrum angle of the angelica dahurica medicinal material, imperatorin is selected as a reference peak, and the relative retention time and the relative peak area of the characteristic peak are calculated, so that the accuracy of quantification of a plurality of characteristic components can be improved.
Drawings
FIG. 1 is a UPLC chromatogram of radix Angelicae Dahuricae under different extraction solvent conditions;
FIG. 2 is a UPLC chromatogram of radix Angelicae Dahuricae under different extraction conditions;
FIG. 3 is a UPLC chromatogram of radix Angelicae Dahuricae under different extraction time conditions;
FIG. 4 is a full-wave scanned 3D UPLC chromatogram of radix Angelicae Dahuricae;
FIG. 5 is a UPLC chromatogram of radix Angelicae Dahuricae under the wavelength conditions of 300nm and 254 nm;
FIG. 6 is a UPLC chromatogram of radix Angelicae Dahuricae under different water phase conditions for mobile phase;
FIG. 7 is a UPLC chromatogram of radix Angelicae Dahuricae under different acetic acid concentrations in mobile phase;
fig. 8 is a UPLC control spectrum of radix angelicae, wherein peak 5: hydrated oxypeucedanin; peak 6: 1, angelica sinensis; peak 8: 8-methoxypsoralen; peak 9: bergamoside lactone; peak 11: white angelica root; peak 12: oxidizing decursin; peak 13: imperatorin; peak 14: phellopterin; peak 15: isoimperatorin;
FIG. 9 is a UPLC characteristic spectrum overlay of 17 batches of radix Angelicae Dahuricae medicinal materials;
FIG. 10 is an identification chart of chromatographic peaks of radix Angelicae Dahuricae;
FIG. 11 is a specificity investigation chromatogram constructed by UPLC contrast map of radix Angelicae Dahuricae;
FIG. 12 is an integrity inspection chromatogram constructed by UPLC contrast map of radix Angelicae Dahuricae;
FIG. 13 is a UPLC chromatogram of a radix Angelicae Dahuricae material to be tested.
Detailed Description
The method for constructing the UPLC characteristic spectrum of the angelica dahurica medicinal material and the detection method thereof are further described in detail with reference to specific embodiments.
Example 1:
the embodiment is a construction method of a UPLC characteristic spectrum of a radix angelicae medicinal material.
1. Apparatus and materials
Waters H-class ultra-high performance liquid chromatograph, Waters PDA detector, Empower workstation; agilent SB C18Chromatography column (100 mm. times.2.1 mm, 1.8 μm); ACQUITY UPLC I-Class ultra-high performance liquid phase flight time high resolution mass spectrum combined systemThe data processing system is a MarkerLynx 4.1 workstation; mettler XP26 parts per million (Mettler Toledo, Switzerland); Milli-Q ultrapure water purification systems (Millipore, USA); model KQ5500DE ultrasonic cleaner (ultrasonic instruments ltd, kunshan). Methanol and acetonitrile are chromatographically pure (Merk); the water is Mili-Q purified water; the rest reagents are analytically pure.
Imperatorin (batch No. 1108206) -201616) and isoimperatorin (batch No. 110827-201611) are provided by the China food and drug testing institute; byakangelicin (batch No. wkq16031401), hydrated oxidized decursin (batch No. wkq17062205), phellopterin (batch No. wkq17101106) are supplied by Vickers Biotech, Inc., Sichuan province; 8-methoxypsoralen (batch: Whar0912) and oxypsoralen (batch: B21471-20mg) were provided by Nanjing Yuanbao pharmaceutical science and technology Co., Ltd; the bergamoolide (batch No. C1420110) is provided by Shanghai Aladdin Biotechnology Ltd; white Angelica sinensis (batch: PO1030SA13) was provided by Shanghai-derived leaf Biotech Co., Ltd; the radix Angelicae Dahuricae is respectively collected from Hebei, Henan, Anhui, and Shandong areas of main producing area, and identified as dry root of Angelica dahurica Angelica Dahurica (Fisch. ex Hoffm.) Benth.et hook.f. of Umbelliferae by senior pharmaceutical engineers of Guangdong-one pharmaceutical company Limited in east China, and the information of the medicinal materials is shown in Table 1.
TABLE 1 information table of radix Angelicae Dahuricae
Evaluation software: the 'Chinese medicine chromatogram characteristic spectrum similarity evaluation system' 2012.0 edition (national pharmacopoeia committee).
2. Chromatographic conditions and preparation of test solutions
2.1 chromatographic conditions: with Agilent SB C18(100 mm. times.2.1 mm, 1.8 μm) as a column, and a flow rate of 0.35 mL/min; the column temperature was 35 ℃; detecting a wavelength of300 nm; gradient elution was carried out using acetonitrile (A) -0.1 vol% aqueous acetic acid (B) as a mobile phase as specified in Table 2 below.
TABLE 2 gradient elution Table
2.2 preparation of reference solutions: accurately weighing appropriate amount of imperatorin reference substance, isoimperatorin reference substance and byak-angelicin reference substance, adding methanol to obtain mixed solution containing 10 ug of imperatorin, 1 ug of isoimperatorin and 15 ug of byak-angelicin per 1mL, and shaking.
2.3 preparation of test solution: taking a proper amount of radix angelicae powder, grinding, sieving by a third sieve, taking about 0.5g, precisely weighing, placing in a conical flask with a plug, precisely adding 20mL of dilute ethanol (ethanol water solution with volume concentration of 45-55%), weighing, ultrasonically treating (300W, 50kHz) for 30min, cooling, weighing again, supplementing the lost weight with dilute ethanol, shaking uniformly, filtering, and taking a subsequent filtrate to obtain the traditional Chinese medicine composition.
2.4 preparation of control solutions: taking a proper amount of imperatorin, isoimperatorin, byak-angelicin, hydrated oxypeucedanin, 8-methoxypsoralen, bergaptide, byak-angelicin, oxypeucedanin and phellopterin reference substances, putting the reference substances into a 25mL measuring flask, adding methanol to a constant volume to scale, and preparing a reference substance solution with the mass concentration of 10, 1, 15, 101, 107, 105, 108, 110 and 115 mg/L.
2.5 measurement method
Precisely sucking 1 μ L of each of the reference solution and the sample solution, injecting into a super high liquid chromatograph, and measuring.
2.6 Mass Spectrometry conditions: detecting a positive ion mode of the electrospray ion source; capillary voltage: 3000V; taper hole voltage: 35V; ion source temperature: 100 ℃; desolventizing gas temperature: 300 ℃; desolventizing air flow rate: 800L/h; the mass scanning range m/z is 150-1500.
3. Examination of preparation method of test solution
3.1 solvent extraction study
Taking 0.5g of angelica dahurica medicinal material powder (passing through a No. three sieve) (S1), weighing 5 parts in parallel, precisely, placing the powder into a conical flask with a plug, precisely adding 20mL of methanol, 50% methanol aqueous solution with volume concentration, diluted ethanol, ethanol and water respectively, weighing, carrying out ultrasonic treatment (power 300W and frequency 50KHz) for 30 minutes, cooling, weighing again, complementing the weight with methanol, 50% methanol aqueous solution with volume concentration, diluted ethanol, ethanol and water respectively, shaking up, filtering, and taking the subsequent filtrate to obtain the medicine. Sample introduction and measurement are carried out according to the chromatographic conditions under the term of '2.1', and the results are shown in Table 3.
TABLE 3 investigation results of different extraction solvents
The result is shown in fig. 1, different extraction solvents are adopted, the number of chromatographic peaks is obviously different, the information content of chromatographic peaks obtained by extracting with methanol, methanol aqueous solution with volume concentration of 50% and dilute ethanol is equivalent, the solvent extraction capacity, the solution stability and the solvent configuration safety are comprehensively considered, and the dilute ethanol is a more suitable solvent.
3.2 examination of extraction methods
The influence of different extraction modes of the characteristic spectrums of the angelica dahurica medicinal material is respectively inspected, and two extraction modes of ultrasonic extraction and reflux extraction are selected for comparison in the research.
Taking radix angelicae powder (passing through a third sieve) (S1) about 0.5g, weighing 2 parts in parallel, precisely weighing, placing in a conical flask with a plug, precisely adding 20mL of diluted ethanol, weighing, respectively performing ultrasonic treatment (power 300W and frequency 50KHz) for 30 minutes, and heating and refluxing for 30 minutes. Cooling, weighing, adding diluted ethanol, shaking, filtering, and collecting filtrate. Sample introduction and measurement were carried out under the chromatographic conditions of "2.1" item, and the results are shown in Table 4.
TABLE 4 examination results of different extraction modes
As shown in FIG. 2, the chromatograms obtained by the two extraction methods of ultrasonic treatment and heating reflux are not different from each other, and the ultrasonic treatment extraction method is adopted in consideration of the simplicity of the operation.
3.3 extraction time study
Weighing radix Angelicae Dahuricae powder (sieved with No. three sieve) (S1) about 0.5g, weighing in parallel 3 parts, placing in a conical flask with a plug, adding diluted ethanol 20ml precisely, weighing, performing ultrasonic treatment (power 300W, frequency 50KHz) for 20, 30 and 40 minutes respectively, cooling, weighing again, supplementing the lost weight with diluted ethanol, shaking, filtering, and collecting the filtrate. Sample introduction and determination were carried out under the chromatographic conditions of "2.1" item, and the results are shown in Table 5.
TABLE 5 examination results of different extraction modes
The results are shown in fig. 3, the above extraction time setting has little effect on the chromatogram, and ultrasound is selected for 30 minutes in order to ensure complete extraction.
4. Chromatographic condition optimization
4.1 determination of the detection wavelength
Weighing radix Angelicae Dahuricae powder (sieved with No. three sieve) (S1) about 0.5g, precisely weighing, placing in a conical flask with a plug, precisely adding 20mL of diluted ethanol, weighing, ultrasonically treating (300W, 50kHz) for 30min, cooling, weighing again, supplementing the weight loss with diluted ethanol, shaking, filtering, and collecting the filtrate. Elution was performed according to the gradient of table 6 below, and the absorption spectrum was recorded in the range of 190-400 nm.
TABLE 6
As shown in FIGS. 4 and 5, the experimental results showed that the absorption of the chromatographic peaks at wavelengths of 300nm and 254nm was good, and that 300nm was used in consideration of the stability of the baseline.
4.2 selection of the Mobile phase
Taking the same sample solution, respectively inspecting mobile phase acetonitrile-water, acetonitrile-phosphoric acid water solution with volume concentration of 0.1%, acetonitrile-formic acid water solution with volume concentration of 0.1% and acetonitrile-acetic acid water solution with volume concentration of 0.1%, and acetonitrile-acetic acid water solution with volume concentration of 0.05% and acetonitrile-acetic acid water solution with volume concentration of 0.1% as an elution system, and carrying out sample injection analysis according to the chromatographic condition of 2.1.
As shown in fig. 6 and 7, the results of the experiments showed that the acetic acid peak at an acetonitrile-volume concentration of 0.1% had a relatively large amount of information, the separation effect of each peak was good, and the peak shape of each component was good, so that an acetic acid aqueous solution system at an acetonitrile-volume concentration of 0.1% was used.
5. Establishment of characteristic spectrum of angelica dahurica medicinal material
5.1 taking 17 batches of angelica dahurica medicinal materials, preparing a test solution according to the item 2.3, and carrying out sample injection determination according to the chromatographic condition under the item 2.1.
5.2 determining the common peak of the characteristic spectrum of the angelica dahurica medicinal material: results of 17 batches of radix angelicae medicinal material UPLC spectra are analyzed by adopting a Chinese medicine chromatogram characteristic spectrum similarity evaluation system (2012.0 version) recommended by the State pharmacopoeia Committee, and 16 common peaks are determined in total. Of the 16 common peaks, the 13 th peak (imperatorin) has good separation degree, symmetrical peak shape and large peak area, and is one of the main components of radix angelicae playing roles of relieving fever, easing pain and resisting inflammation, so the 13 th peak (imperatorin) is taken as a reference peak (namely S peak), the reference map is shown in figure 8, the relative retention time and the relative peak area of each common peak are respectively calculated, and the results are shown in table 7 and table 8. As can be seen from the relative retention times of the common peaks of the samples (Table 7), the relative retention times of the common peaks of the samples are very close, and the RSD is between 0.06% and 2.76%, indicating the accuracy of the selection of the common peaks. As can be seen from the relative peak areas of the common peaks of the samples (Table 8), the peak areas of the common peaks of different samples have a large difference, which indicates that the content of the common peaks of radix angelicae in different producing areas has a certain difference.
TABLE 7 relative Retention time values for Angelica dahurica
TABLE 8 relative peak area values of Angelica dahurica
The results show that: the characteristic spectrum of the test sample should present 16 characteristic peaks, and should correspond to 16 characteristic peaks in the chromatogram of the reference substance of the reference medicinal material; the peak 13 corresponding to the imperatorin reference peak is an S peak, the relative retention time of each characteristic peak and the S peak is calculated, the relative retention time is within +/-10% of a specified value, and the specified value is as follows: peak 1: 0.19, peak 2: 0.26, peak 3: 0.35, peak 4: 0.43, peak 5: 0.49, Peak 6: 0.51, peak 7: 0.58, Peak 8: 0.61, peak 9: 0.67, peak 10: 0.71, peak 11: 0.79, peak 12: 0.81, peak 13: 1. peak 14: 1.05, peak 15: 1.11, peak 16: 1.12, calculating the relative peak area of each characteristic peak and the peak 13, wherein the relative peak area is in a specified range: peak 1: 0.01 to 0.08, Peak 2: 0.01 to 0.03, Peak 3: 0.01 to 0.03, Peak 4: 0.01 to 0.04, Peak 5: 0.18 to 1.15, Peak 6: 0.01 to 0.21, Peak 7: 0.01 to 0.05, Peak 8: 0.05 to 0.21, Peak 9: 0.07-0.22, Peak 10: 0.01 to 0.12, Peak 11: 0.12 to 0.40, peak 12: 0.38-1.24, Peak 13: 1. peak 14: 0.22-0.45, Peak 15: 0.23 to 0.50, peak 16: 0.02 to 0.04.
5.3 evaluating the similarity of the characteristic spectrums of the angelica dahurica medicinal materials: the method is characterized in that a Chinese medicine chromatogram characteristic map similarity evaluation system (2012.0 version) recommended by the State pharmacopoeia Commission is adopted to superpose characteristic maps of angelica dahurica medicinal material samples of No. S1-S17, the consistency of the similarity of chromatographic peak is inspected, the similarity evaluation is carried out, the result is shown in a figure 9 and a table 9, the result shows that the similarity of the angelica dahurica medicinal material samples of 4 different producing areas is more than 0.9, the similarity of the sample characteristic map and a reference map is higher, 16 common peaks can stably appear in each sample, and the goodness of fit is better, so the map can be used as the characteristic map of the angelica dahurica medicinal material.
TABLE 9 similarity of different samples of Angelica dahurica
6. Identification of common peaks
And respectively detecting the test solution and the reference solution by adopting the chromatographic condition under the item of 2.1 and the mass spectrum condition under the item of 2.6. The chromatographic peak retention behavior, accurate molecular weight and comparison with a reference substance of the compounds determine 9 compounds in total. According to the retention time, hydrated oxypeucedanin (peak 5), byak-angelicin (peak 6), 8-methoxypsoralen (peak 8), bergapten (peak 9), byak-angelicin (peak 11), oxypeucedanin (peak 12), imperatorin (peak 13, S), phellopterin (peak 14) and isoimperatorin (peak 15) are respectively. The chromatogram peak superposition of the test solution and 9 reference solutions is shown in FIG. 10, and the related information of each chromatogram peak is shown in Table 10.
7. Methodology investigation
(1) Specialization inspection
Taking radix Angelicae Dahuricae powder (sieved with No. three sieve) (S1), preparing into sample solution and blank solvent (diluted ethanol) according to the method determined under item "2.3", preparing into reference solution according to the method determined under item "2.2", introducing sample under the chromatographic condition of item "2.1", measuring, and recording chromatogram.
The experimental results are as follows: as shown in fig. 11, 16 chromatographic peaks of the characteristic spectrum of the angelica dahurica medicinal material are not interfered by a negative blank solvent, wherein the retention time of characteristic spectrum byak-angelicin, imperatorin and isoimperatorin peaks in the medicinal material is consistent with that of a reference solution.
(2) Integrity survey
Taking radix Angelicae Dahuricae powder (sieved with No. three sieve) (S1), and preparing into test solution according to the method determined under item "2.3". Chromatographic peaks were analyzed under chromatographic conditions under "2.1" with the final gradient maintained and the elution time extended to 2 times the total time, as shown in table 11 below:
TABLE 11 gradient elution procedure for integral inspection of characteristic spectrum of radix Angelicae Dahuricae medicinal material
The experimental results are shown in fig. 12: no obvious chromatographic peak is generated after 18 minutes, and the chromatographic peak basically meets the maximum information content principle under the current chromatographic conditions.
(3) Precision survey
Taking radix Angelicae Dahuricae powder (sieved by No. three sieves) (S1), preparing into test solution according to the method determined under item "2.3", continuously feeding sample for 6 times according to the chromatographic condition under item "2.1", measuring, taking the imperatorin chromatographic peak as reference peak S, calculating the relative retention time of peak 1-peak 16, and calculating RSD value.
The experimental results are as follows: in precision investigation of continuous sample injection for 6 times, the RSD of 16 chromatographic peaks of the angelica dahurica medicinal material relative to retention time is less than 2%, and the RSD of the relative peak area is less than 2%, which indicates that the precision of the characteristic spectrum method is good.
(4) Stability survey
Taking radix Angelicae Dahuricae powder (sieved by a third sieve) (S1), preparing into test solution according to the method determined under item "2.3", standing at room temperature according to the chromatographic condition under item "2.1", sampling and measuring at 0h, 2h, 4h, 6h, 8h and 12h, calculating the relative retention time and relative peak area of peaks 1-16 by using imperatorin chromatographic peak as reference peak S, and calculating RSD value.
The experimental results are as follows: the RSD of 16 chromatographic peaks of the angelica dahurica medicinal material relative to the retention time is less than 2 percent, and the RSD of the relative peak area is less than 2 percent, which shows that the characteristic spectrum method has good repeatability.
(5) Repeatability survey
Taking radix Angelicae Dahuricae powder (sieved by a third sieve) (S1), making into test solution by 6 parts in parallel according to the method determined under the item "2.3", performing sample injection measurement according to the chromatographic condition under the item "2.1", taking an imperatorin chromatographic peak as a reference peak S, calculating the relative retention time and the relative peak area of peaks 1-16, and calculating the RSD value.
The experimental results are as follows: the RSD of 16 chromatographic peaks of the angelica dahurica medicinal material relative to the retention time is less than 2 percent, and the RSD of the relative peak area is less than 2 percent, which shows that the characteristic spectrum method has good repeatability.
Example 2
The embodiment is a detection method of a radix angelicae medicinal material, which comprises the following steps:
1. chromatographic conditions
With Agilent SB C18(100 mm. times.2.1 mm, 1.8 μm) as a column, and a flow rate of 0.35 mL/min; the column temperature was 35 ℃; the detection wavelength is 300 nm; gradient elution was carried out using acetonitrile (A) -0.1 vol% aqueous acetic acid (B) as a mobile phase as specified in Table 2 below.
TABLE 2 gradient elution Table
2. Preparing a sample solution to be tested: grinding radix Angelicae Dahuricae to be measured (S18), sieving with a third sieve, precisely weighing about 0.5g, placing in a conical flask with a plug, precisely adding 20mL of dilute ethanol (45-55% ethanol water solution), weighing, ultrasonically treating (300W, 50kHz) for 30min, cooling, weighing, supplementing the weight loss with dilute ethanol, shaking, filtering, and collecting the filtrate.
3. And (3) determination: precisely absorbing 1 mu L of sample solution to be detected, injecting the sample solution into an ultra-high performance liquid chromatograph, and measuring according to the chromatographic condition of 1 to obtain the product.
The result is shown in fig. 13, and as can be seen by comparing the characteristic spectrum of the angelica dahurica reference drug material established in example 1 (fig. 8), the result is shown in fig. 13, and the characteristic spectrum of the test sample shows 16 characteristic peaks, which correspond to the 16 characteristic peaks in the characteristic spectrum of the angelica dahurica reference drug material; and calculating the relative retention time and the relative peak area of each characteristic peak and the S peak, wherein the relative retention time and the relative peak area are within a specified range, and the similarity is 0.94 and is more than 0.90, which indicates that the medicinal material meets the quality requirement.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A method for constructing a UPLC characteristic spectrum of a radix angelicae medicinal material is characterized by comprising the following steps:
preparation of reference solutions: weighing imperatorin reference substance, and dissolving with solvent to obtain reference substance solution;
preparation of a test solution: weighing radix Angelicae Dahuricae, adding extraction solvent, performing ultrasonic or reflux extraction, filtering, and collecting filtrate to obtain the sample solution; the extraction solvent is methanol, methanol water solution with volume concentration of 45-55%, ethanol or ethanol water solution with volume concentration of 45-55%;
establishing a characteristic spectrum: respectively sucking the reference substance solution and the test solution, injecting the reference substance solution and the test solution into an ultra-high performance liquid chromatograph, and measuring to obtain the characteristic map; the characteristic map comprises characteristic peaks of hydrated oxypeucedanin, byak-angelicin, 8-methoxypsoralen, bergapten, byak-angelicin, oxypeucedanin, imperatorin, phellopterin and isoimperatorin;
wherein the ultra-high performance liquid chromatography adopts the following elution conditions: acetonitrile is taken as a mobile phase A, and acetic acid water solution with volume concentration of 0.05-0.15% is taken as a mobile phase B; gradient elution is adopted;
the gradient elution method comprises the following steps:
the volume percent of the mobile phase A is increased to 28 percent from 15 percent and the volume percent of the mobile phase B is reduced to 72 percent from 85 percent in 0-5.5 min;
5.5-7min, the volume percent of the mobile phase A is increased from 28% to 40%, and the volume percent of the mobile phase B is decreased from 72% to 60%;
7-9.5min, and keeping the volume percent of the mobile phase A at 40% and the volume percent of the mobile phase B at 60%;
9.5-15.5min, the volume percent of the mobile phase A is increased from 40% to 65%, and the volume percent of the mobile phase B is decreased from 60% to 35%;
15.5-15.51min, the volume percent of the mobile phase A is reduced from 65% to 15%, and the volume percent of the mobile phase B is increased from 35% to 85%;
15.51-18min, keeping the volume percentage of the mobile phase A at 15% and the volume percentage of the mobile phase B at 85%.
2. The method for constructing the UPLC feature map of the radix angelicae dahuricae medicinal material according to claim 1, wherein the extraction solvent is an ethanol aqueous solution with a volume concentration of 45-55%.
3. The method for constructing the UPLC feature map of the radix angelicae dahuricae medicinal material according to claim 1, wherein the extraction solvent is an ethanol aqueous solution with a volume concentration of 45-55%, and the amount of the extraction solvent is 10-50mL per 0.5g of the radix angelicae dahuricae medicinal material; the extraction time is 20-40 min.
4. The UPLC feature spectrum construction method of radix angelicae medicinal material according to any one of claims 1-3, wherein the conditions of the ultra-high performance liquid chromatography are as follows: octadecylsilane chemically bonded silica is used as a filler for the chromatographic column; the flow rate of the mobile phase is 0.25-0.4 mL/min; the column temperature is 30-40 ℃; the detection wavelength is 300nm or 254 nm.
5. The method for constructing the UPLC feature map of the radix angelicae dahuricae medicinal material according to any one of claims 1-3, wherein the feature map comprises 16 feature peaks, the chromatographic peak of 13 imperatorin is taken as a reference peak S peak, the relative retention time of each feature peak is within ± 10% of a specified value, and the specified value is as follows: peak 1: 0.19, peak 2: 0.26, peak 3: 0.35, peak 4: 0.43, peak 5: 0.49, Peak 6: 0.51, peak 7: 0.58, Peak 8: 0.61, peak 9: 0.67, peak 10: 0.71, peak 11: 0.79, peak 12: 0.81, peak 13: 1. peak 14: 1.05, peak 15: 1.11, peak 16: 1.12.
6. the detection method of the radix angelicae medicinal material is characterized by comprising the following steps of:
preparing a sample solution to be tested: weighing radix Angelicae Dahuricae to be tested, adding extraction solvent, performing ultrasonic or reflux extraction, filtering, and collecting filtrate to obtain the sample solution; the extraction solvent is methanol, methanol water solution with volume concentration of 45-55%, ethanol or ethanol water solution with volume concentration of 45-55%;
and (3) detection: absorbing the sample solution to be detected, injecting the sample solution into an ultra-high performance liquid chromatograph, and detecting;
wherein the ultra-high performance liquid chromatography adopts the following elution conditions: acetonitrile is taken as a mobile phase A, and acetic acid water solution with volume concentration of 0.05-0.15% is taken as a mobile phase B; gradient elution is adopted;
the gradient elution method comprises the following steps:
the volume percent of the mobile phase A is increased to 28 percent from 15 percent and the volume percent of the mobile phase B is reduced to 72 percent from 85 percent in 0-5.5 min;
5.5-7min, the volume percent of the mobile phase A is increased from 28% to 40%, and the volume percent of the mobile phase B is decreased from 72% to 60%;
7-9.5min, and keeping the volume percent of the mobile phase A at 40% and the volume percent of the mobile phase B at 60%;
9.5-15.5min, the volume percent of the mobile phase A is increased from 40% to 65%, and the volume percent of the mobile phase B is decreased from 60% to 35%;
15.5-15.51min, the volume percentage of the mobile phase A is reduced from 65% to 15%, and the volume percentage of the mobile phase B is reduced from 35% to 85%;
15.51-18min, keeping the volume percentage of the mobile phase A at 15% and the volume percentage of the mobile phase B at 85%.
7. The method for detecting radix angelicae dahuricae according to claim 6, wherein the extraction solvent is an ethanol aqueous solution with a volume concentration of 45-55%.
8. The method for detecting radix angelicae dahuricae according to claim 6, wherein the extraction solvent is 45-55% ethanol water solution by volume, and the amount of the extraction solvent is 10-50mL for every 0.5g of radix angelicae dahuricae.
9. The method for detecting radix angelicae dahuricae according to claim 6, wherein the extraction time is 20-40 min.
10. The detection method of radix angelicae dahuricae medicinal material according to any one of claims 6-9, wherein the conditions of the ultra high performance liquid chromatography are as follows: octadecylsilane chemically bonded silica is used as a filler for the chromatographic column; the flow rate of the mobile phase is 0.25-0.4 mL/min; the column temperature is 30-40 ℃; the detection wavelength is 300nm or 254 nm.
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